Extraction method

ABSTRACT

There is disclosed a method for treating a source of azadirachtin: (a) dissolving said sample in an organic polar solvent; (b) combining the solution of step (a) with an organic non-polar solvent to form a single phase containing the said solvents and dissolved sample; (c) treating said single phase of step (b) with an aqueous salt solution to form an aqueous phase and a phase containing the organic non-polar solvent; and (d) recovering one or other or both of the aqueous phase and the phase containing the organic polar solvent.

This application is a 371 of PCT/GB01/04590 filed on Oct. 15, 2001.

This invention relates to a method of treating an impure sample ofazadirachtin isolated from a natural source to increase its purity.

Azadirachtin A (normally simply referred to as azadirachtin) is presentthroughout the Neem tree (Azadirachta Indica) and in significantquantities of up to 0.8% in the seed and is well known for itspotentially wide ranging beneficial properties including insecticidalproperties. Azadirachtin has been found to occur in several other plantswhich include the Chinaberry Tree and the Babool Bush. In addition,several hundred compounds have been identified in the Neem Tree, manyoccurring in the seed including the 12 main triterpenoids which includenimbin, salannin and 3-tigloylazadirachtol (also known as azadirachtinB). Several of these compounds have been shown to have beneficialpharmaceutical and insecticidal properties. The table at the end of thisdescription contains a list of the phytochemicals of Azadirachta Indicaand their location (bark, seed, etc.)

Methods are known in which the neem seed (or other azadirachtin source)is processed, including solvent extraction steps to obtain a crude solidextract containing the azadirachtin; typically such known processes(e.g. U.S. Pat. No. 5,856,526) result in a product containing normallyup to 15% azadirachtin and occasionally 25–30%. In order to isolate pureazadirachtin A (e.g. above about 90% purity), expensive chromatographicmethods are normally used. As a result azadirachtin with a purity of 80%currently retails for about US$ 85,000 per gram.

The present invention provides a novel solvent extraction processwhereby the purity of a crude sample of azadirachtin can be increased,potentially up to a purity in excess of 90%, without the need to resortto chromatographic methods. The process may also enable the otherpotentially valuable by-products such as nimbin, salannin and geduninassociated with the natural sources of azadirachtin to be concentratedand isolated.

According to a first aspect of the present invention, there is provideda method for treating a source of azadirachtin:

(a) dissolving said sample in an organic polar solvent;

(b) combining the solution of step (a) with an organic non-polar solventto form a single phase containing the said solvents and dissolvedsample;

(c) treating said single phase of step (b) with an aqueous salt solutionto form an aqueous phase and a phase containing the organic non-polarsolvent; and

(d) recovering one or other or both of the aqueous phase and the phasecontaining the organic polar solvent.

When, as will normally be the case, the aqueous phase is recovered, itmay be treated to isolate an azadirachtin-containing componenttherefrom. Alternatively, as described in more detail below, it may bepooled with subsequent washings of the recycled organic non-polar phaseand then treated to separate the azadirachtin-containing componenttherefrom.

It has been found that, when a crude sample of azadirachtin is treatedin accordance with this method aspect of the invention, an equilibriumis established between the organic and aqueous phases in which theazadirachtin A, D, E and I and, it is believed, also the geduninpreferentially accumulate as either a syrup-like or solid material(depending on the stage of purification) which is found in the aqueouslayer or phase of step (c). The azadirachtin B, nimbin and salanninpreferentially accumulate in the organic non-polar layer. This providesa means whereby the various compounds in the starting sample can beseparated from each other and enriched.

The method of the present invention may be used to isolate any one ormore of the different components of natural azadirachtin sources such asthe neem seed. Although this would normally include isolation of theazadirachtin A, which is an extremely valuable natural product when inthe pure form, it is not necessarily the case that the azadirachtin Awill be isolated in all embodiments of the invention; thus, in someembodiments of the invention the method may be carried out to isolateother components of the neem seed.

In preferred embodiments of the method of this aspect of the invention,the non-polar organic phase in step (c) is recovered and subjected againto the treatment of step (c). This treatment of the non-polar organicphase with the aqueous salt solution may be repeated several times untilno more solid matter is observed accumulating in the aqueous phase. Thismay require, for example, up to 10 cycles, typically up to 5 cycles. Theazadirachtin-containing component may be isolated from the aqueous phaseat the end of each cycle and combined; alternatively, the recoveredaqueous phases from each cycle may be pooled and, if desired at thisstage, the azadirachtin-containing component may be isolated. Startingwith a 30% purity sample, and after cycling in this way, the solidproduct obtained may typically contain about 45% azadirachtin A.

Where the starting sample has a low concentration of azadirachtin (e.g.5–10%), it may be desirable, rather than recycling the organic non-polarphase, to isolate the solid azadirachtin-containing sample from theaqueous phase and recycle this through process steps (a)–(c). This maybe carried out several times until the concentration of azadirachtin inthe solid material reaches a concentration of about 40%.

The azadirachtin-containing sample which is subjected to the method ofthe present invention may be one which contains at least 10%azadirachtin, and thus results from a preliminary processing of thenatural source (which may contain varying amounts of the variouscompounds of interest depending on the quality of the seed or othersource) in order to increase the azadirachtin content to that level. Thebetter the purity of the starting azadirachtin, the fewer cycles arerequired to achieve a high purity material. Good results may be obtainedusing, as the starting material for the process of the invention, anazadirachtin-containing sample in which the azadirachtin content is ofthe order of 25–30%. As an alternative, it has been found that theprocess of the present invention may be used to purify a sample takendirect from the neem seed (or other primary source of azadirachtin),i.e. with no preliminary processing. This provides a valuablealternative to the use of the conventional preliminary processing stepsof a primary source of azadirachtin.

Where a conventional initial extraction method is used, the precisenature of this is not crucial to the present invention, although it isnoted that different initial extraction methods result in differentconcentrations of the various compounds of interest in the resultingproduct. An example of a preliminary processing method has the followingsteps:

1. Preparation of the seed by air drying in the shade and separation ofthe seed from the pulp. It is important not to dry in the full sun lightas this may cause the azadirachtin to break down.

2. Grinding of the dried seed with a solvent to remove the oil. Neemseed can contain up to 50% oil which must be removed using a suitablesolvent such as hexane. The neem oil is separated from the solvent whichis recovered and the oil retained for sale, further processing orformulation.

3. Soaking of the seed cake in a polar solvent such as methanol for anextended period of up to 24 hours without the application of heat andwith slow stirring. The use of heat tends to cause the break down of theazadirachtin. Repeat extraction three times to maximise the amount ofcompounds in the extract.

4. Separation of the methanol liquid containing the useful compoundsincluding azadirachtin from the seed cake. Separation may be by anysuitable means including filtration under vacuum.

5. Remove up to 80% of the methanol under reduced pressure to produce athick syrup. Water is then added to the syrup.

6. Add dichloromethane in the ratio of about 4:3 parts of syrup mixture.Stir for up to 45 minutes then allow to settle into two layers. Thelower dichloromethane layer is removed for further processing and theaqueous layer is treated with the same volume of dichloromethane in theratio 4:3. The extraction is completed at least 3 times to ensurecomplete removal of azadirachtin from the syrup.

7. All the dichloromethane layers are combined and dried with a suitabledrying agent such as magnesium sulphate. The drying agent is removed byfiltration and dichloromethane recovered. The extract is treated withtwice its weight of hexane to form a precipitate, which is recovered byfiltering. The precipitate then typically contains approximately 5%–15%azadirachtin.

In step (a) of the method of the first aspect of this invention, thesample is dissolved in an organic polar solvent. Examples of suitablesolvents include acetone, methanol and ethanol or mixtures thereof, withmethanol or acetone being preferred. The amount of organic polar solventused should be sufficient to dissolve the sample, but otherwise is notcritical. An amount of 200 ml of solvent per 100 g of sample has beenfound to be satisfactory where the solvent is acetone or methanol.Typically, the material is added to the solvent and stirred for asufficient time (e.g. 30 minutes) to dissolve the material. Any materialwhich is insoluble may be removed, for example by filtration.

The solution of step (a) is then combined with an organic non-polarsolvent to form a single phase which contains the said solvents and thedissolved material. The organic non-polar solvent used in this step ispreferably a halogenated hydrocarbon, such as carbon tetrachloride,chloroform or dichloromethane. However, other non-polar solvents, suchas toluene are also operable. The presently preferred non-polar solventis carbon tetrachloride. An amount of the organic non-polar solvent atleast equal to the amount of the organic polar solvent used in step (a)may be employed. Ratios of organic polar solvent to non-polar organicsolvent up to 1:20 are operable, but preferred is about 1:2.

Typically, the single phase formed in step (b) is a brown to dark browncolour, depending on the purity of the starting material.

In step (c), the single phase of step (b) is treated with an aqueoussalt solution. On the laboratory scale, this step may be carried out ina separating funnel to facilitate separation of the phases. Thesolutions should be thoroughly mixed and then allowed to separate toform two layers, an aqueous layer and a non-polar solvent layer below. Asolid or syrupy material (depending upon the purity of the initialsample) separates in the aqueous layer.

Preferred salts are those which form a solution which is approximatelyneutral in pH (about 6.5–7.5) since basic or acid salt solutions maytend to cause the azadirachtin or other components to decompose.Preferred salts are the water soluble alkali metal and alkaline earthmetal halides, with sodium chloride being particularly preferred. Thesalt solution is preferably non-saturated. A solution containing betweenabout 5% and saturated is satisfactory, with a concentration of about10% being optimum.

The volume of salt solution relative to the volume of the organic phaseresulting from step (b) may range from 1:1 to 15:1. Preferred is a ratioof 2:1 by volume. The mixture should be thoroughly agitated or mixed andthen allowed to stand long enough to achieve separation, for example fora period of time of the order of 30 minutes, to produce two layers. Theupper layer is the aqueous salt layer and the lower layer is the organicnon-polar layer. The layers are separated and it is preferred that theorganic non-polar layer is then subjected to further cycles of treatmentwith salt solution until no more solid matter is observed to accumulatein the aqueous-phase, as described above. In the first salt solutionwashing, a dark brown syrupy substance appears in the aqueous phase.With repeated cycles this becomes whiter and less syrupy and the volumedecreases.

The azadirachtin-containing component may be separated from the aqueousphase each cycle or the aqueous salt solution washings may be retainedand combined and the solids recovered, for example by filtration. Therecovered solid is normally a yellow/white powder and is enriched inazadirachtin relative to the starting material, for example up to about45% azadirachtin.

The aqueous phase from each cycle, after removal of theazadirachtin-containing component may be recovered and retained forfurther processing to recover any water-soluble compounds of interesttherefrom.

The organic non-polar phase may be retained and treated further. Thismaterial contains 3-tigloylazadirachtol (sometimes known as Aza B) andalso a number of the other triterpenoids such as nimbin and salannin, aswell as some other compounds.

In order to increase the purity of the azadirachtin-containing componentseparated from the aqueous phase still further, the recovered solid maybe subjected to at least one further round of the process of the presentinvention. The same or different solvents may be used in the second andany further rounds. For example, in one experiment, good results havebeen obtained where, in the first round, methanol is used as the polarsolvent, and, in the second round, acetone is used as the polar solvent.

As mentioned previously, the azadirachtin-containing component isolatedfrom the aqueous phase in the process of the invention contains thecompounds azadirachtin A, D, H and I. In order to purify this componentfurther, the solid may be dissolved in a first solvent in which thevarious compounds are soluble, for example chloroform or ethyl acetate.The minimum amount to completely dissolve the solid material may beemployed. To this solution is added a second solvent which is matchedwith the first solvent so as to give a precipitate of the dissolvedcompounds other than the azadirachtin A. Thus, where the first solventis chloroform, the second solvent may be ether, for example in the ratioof approximately 1:3. Where the first solvent is ethyl acetate, thesecond solvent may be hexane. The first and second solvents arepreferably low boiling point solvents so that they can be removed at arelatively low temperature which does not decompose any of the valuablecompounds separated. A yellow to white solid is formed which containsmainly azadirachtin D, R and I, together with some azadirachtin A. Thismaterial can be retained for further separation of these triterpenoidsif required. The chloroform/ether layer (or other combination of firstand second solvent), contains the majority of the azadirachtin A fromwhich it may be isolated. For example, the ether may be removed from themixture and the chloroform reduced to approximately 20% of its originalvolume. The solvents may be recovered for reuse. To the concentratedsolution may be added three times the volume of a solvent in which theazadirachtin A is insoluble and of low boiling point such as hexane,pentane or heptane, most preferably hexane to produce a white solidwhich may contain approximately 70% azadirachtin A. The hexane-richfiltrate may be recovered and recirculated.

The organic non-polar layer which may be recovered in step (c) of theprocess of the invention contains mainly the azadirachtin B with someazadirachtin A, salannin, nimbin and other similar compounds. This layermay, for example, be treated to remove at least 50% of the organicsolvent under reduced pressure, and then treated with an equal volume ofhexane. The majority of azadirachtin A and some azadirachtin B isprecipitated, whilst the filtrate contains the majority of theazadirachtin B and other compounds.

At all stages the compounds are partitioned selectively between the twolayers. Repeated reprocessing of the partitioned compounds causes thecompounds to be concentrated giving purities in excess of 60% for theindividual target compounds of interest.

At all stages the various fractions and solvents are recycled orrecovered for reuse so reducing the overall cost of the extraction andpurification.

In accordance with the process of the present invention, largequantities of pure azadirachtin A may be produced, which has hithertoproved impossible. Thus, in accordance with another aspect of theinvention, there is provided a package containing at least 10 g ofazadirachtin A having a purity of at least 50%. Any suitable packagingfor the azadirachtin A product may be employed, such as plastic bags andglass, or plastic tubes. Preferably, the azadirachtin A in the packagehas a purity of at least 70%, preferably at least 90%. The package maycontain at least 100 g of the pure azadirachtin A, preferably at least100 g of azadirachtin A.

The invention will now be illustrated, by way of the followingnon-limiting example.

EXAMPLE

This example describes the methods and techniques used to purifyazadirachtin A and B from a sample of technical azadirachtin with aninitial concentration of 30% azadirachtin A. The example was worked on aweight sample of 200 grams.

Step 1.

Accurately weight 200 of technical grade Azadirachtin, analysis 33%azadirachtin A.

Step 2.

Dissolve in 400 mls of methanol at room temperature 18° C., stir untilcompletely dissolved, which in this example takes about 15 minutes.

Step 3.

When fully dissolved and whilst still stirring 800 mls of carbontetrachloride (ratio of 2:1 to the methanol) was added. The mixture wasstirred for a further 10 minutes.

Step 4.

200 mls of 10% brine was added, and stirred as rapidly as possible usingan overhead stirrer. Solid material was formed in the brine layer. Theliquid was allowed to separate into two layers with the lower layerbeing the carbon tetrachloride layer. The two layers were separatedusing a separating funnel and the brine layer filtered to remove theprecipitate. This precipitate was dried under vacuum until constantweight, usually about 2 hours. The weight obtained was 80 grams. Ananalysis sample was prepared by weighing 19 mg of the precipitate anddissolving in 19 ml acetonitrile (1:1). 100 μl of the solution wasdiluted with 5 ml of hlpc grade water. The sample was analysed by HPLCto give an azadirachtin A content of 56%.

Step 5.

A further 200 ml of the brine solution was added to the separated carbontetrachloride layer, and the liquid rapidly stirred as before. A furtherprecipitate was formed in the brine layer which is recovered aspreviously. The precipitate reduces in volume with each washing and itscolour changes from yellow to white. The cycle is repeated until nofurther precipitate appears. This usually takes 4 to 5 washings. Thecarbon tetrachloride layer was retained for further treatment. Thislayer contained the azadirachtin B. The brine washings contained 21grams of solid having an azadirachtin A content of 56%.

Step 6.

The process was repeated a third time by the addition of a further 200ml of brine to the residual carbon tetrachloride layer of step 5. Thesolid formed was 46 grams and was found to have an azadirachtin Acontent of 69%.

Step 7.

The process was repeated a fourth time by the addition of a further 200ml of brine to the residual carbon tetrachloride layer of step 6. Thesolid formed was 13 grams having an Azadirachtin A content of 58%.

Step 8.

The process was repeated a fifth time with the addition of 200 ml ofbrine to the residual carbon tetrachloride layer of step 7. No furthersolid was yielded. The carbon tetrachloride layer was dried with MgSO₄,filtered, reduced to ⅓ its volume and three times the volume of hexaneadded. A pale yellow solid formed weighing 12.3 grams. Analysis of thissample gave azadirachtin B at 46% and azadirachtin A at 26%.

Step 9.

All the brine water washings were combined and 500 ml of chloroform wasadded. The resultant mixture was stirred or otherwise mixed and thenallowed to separate. The chloroform layer was separated, dried withMgSO₄, and the chloroform recovered to ⅓ volume. Hexane was added givinga solid which was removed by filtration. The solid yielded 15 gramsgiving 53% azadirachtin A content and 7% azadirachtin B.

This was the end of the first “loop”.

Step 10.

The solid from the first four brine washings was added-together to givea total weight of 160 grams. This was dissolved in 320 ml of acetone andstirred until fully dissolved. After 15 minutes, 740 ml CCl₄ was addedand stirred. The resulting solution was washed with 640 ml of 10% brinein 4 parts 160 ml each.

Step 11.

As with the initial addition of brine, solid formed in the brine layerwhich was separated and dried as previously. The first washing gave 28grams azadirachtin A content 83%.

Step 12.

The second washing with 160 ml of brine produced 4 grams of solid havingan Azadirachtin A content of 65%.

Step 13.

The washing was repeated as previously and the solid separated and driedto give a weight of 6.6 grams and an azadirachtin A content of 49%.

Step 14.

The fourth washing gave a syrup rather than a solid. This was dissolvedin a minimum of chloroform and hexane added to give a white solid andyellow syrup. The white solid was filtered to give 18 grams of producthaving an azadirachtin A content of 40% and an azadirachtin B of 30%.The syrup was separated and dried to give a 10 g of a solid product(azadirachtin A 57%).

The total weight recovered at this point was 142 grams.

Step 15.

The samples from steps 11 onwards were combined and dissolved inchloroform to which ether was then added to form a solid. This wasremoved by filtering and drying to yield 28 grams of product.

Step 16.

The ether was removed from the filtrate by vacuum and five times thevolume of hexane added. A white solid rich in azadirachtin A wasobtained (azadirachtin A, 40 g, purity 70%). The filtrate was treated toprecipitate 12 g of azadirachtin B material. The various washings werecombined and 18 g of a precipitate containing azadirachtin A andazadirachtin B was obtained.

The 28 grams of azadirachtin A of over 80% purity from step 15 wasretained and the various fractions with a lower azadirachtin contentwere re-circulated by adding to fresh technical grade material andreprocessed. In this way the concentration of the azadirachtin A wasincreased until it reached the required level. Similarly with theazadirachtin B, this was recycled until the required concentration wasobtained.

Neem seed extract contains many different compounds in addition to theAzadirachtin A and B. These include Azadirachtin D, H, I, Nimbin andsalannin. The process concentrates some of these compounds in the brinelayer and some in the carbon tetrachloride layer allowing theirseparation and purification.

TABLE 1-TIGLOYL-3-ACETYL-11-METHOXY- Bark AZADIRACHTININ1ALPHA-METHOXY-1,2-DIHYDROAZADIRADIONE Seed 1BETA,2BETA-DIEPOXY-AZADIRADIONE Seed 3-ACETYL-7-TIGLOYL-LACTONE-VILASININLeaf 3-DESACETYL-3-CINNAMOYL-AZADIRACHTIN Leaf 3-DEACETYL-SALININ Leaf3-DESACETYLSALANNIN Seed 3-TIGLOYLZAZDIRACHLOL Seed 4-EPINIMBIN Seed4ALPHA, 6ALPHA-DIHYDROXY-A-HOMO- Leaf AZADIRADIONE 6-ACETYL-NIMBANDIOLSeed 6-DESACETYLNIMBINENE Bark 6-DESACETYLNIMBINENE Leaf6-DESACETYLNIMBINENE Seed 6-O-ACETYL-NIMBANDIOL Plant7-ACETYLNEOTRICHILENONE Seed 7-DESACETYL-7-BENZOYL-AZADIRADIONE Seed7-DESACETYL-7-BENZOYLEPOXY-AZADIRADIONE Seed7-DESACETYL-7-HYDROXY-AZADIRADIONE Fruit 7-DESACETYL-GEDUNIN Seed17-BETA-HYDROXYAZADIRDIONE Seed 17-EPIAZADIRADIONE Seed22,23-DIHYDRO-23BETA-METHOXY-AZADIRACHTIN Seed ARACHIDIC-ACID Fruit ASHFruit ASH Leaf AZADIRACHTANIN Leaf AZADIRACHTANIN-A Leaf AZADIRACHTINSeed AZADIRACHTOL Fruit AZADIRADIONE Seed AZADIRONE Seed BEHENIC-ACIDFruit BETA-SITOSTEROL Flower BETA-SITOSTEROL Leaf CALCIUM Fruit CALCIUMLeaf CARBOHYDRATES Fruit CARBOHYDRATES Leaf DESACETYLNIMBIN Stem Bark EOStem Bark EPOXYAZADIRADIONE Seed FAT Fruit FAT Fruit FAT Seed FIBER LeafGEDUNIN Seed HYPEROSIDE Leaf ISOAZADIROLIDE Leaf ISOMARGOSINOLIDE PlantISONIMBINOCINOLIDE Plant ISONIMBINOLIDE Stem Bark ISONIMBOCINOLIDE LeafISONIMOLICINOLIDE Fruit KAEMPFEROL Flower LIGNOCERIC-ACID FruitLINOLEIC-ACID Fruit MAGNESIUM Fruit MAGNESIUM Leaf MARGODUNOLIDE PlantMARGOSINE Stem Bark MARGOSINOLIDE Plant MELDENIN Seed MELIANTRIOL SeedMYRICETIN Flower MYRISTIC-ACID Fruit NIMBAFLAVONE Leaf NIMBANDIOL LeafNIMBANDIOL Stem NIMBIDIN Seed NIMBIDIN Stem Bark NIMBIN Stem BarkNIMBINENE Bark NIMBINENE Leaf NIMBINENE Stem NIMBININ Stem BarkNIMBINONE Stem Bark NIMBIOL Bark NIMBIONE Stem Bark NIMBOCINOLIDE PlantNIMBOCINOME Plant NIMBOLIDE Leaf NIMBOLIN-A Wood NIMBOLIN-B WoodNIMBOSTEROL Stem Bark NIMOCINOL Fruit NIMOLICINOIC-ACID FruitNIMOLICINOL Seed NIMOLINONE Fruit NONACOSANE Flower OLEIC-ACID FruitPALMITIC-ACID Fruit PHOSPHORUS Fruit PHOSPHORUS Leaf PROTEIN FruitPROTEIN Leaf QUERCETIN Flower QUERCETIN Leaf QUERCITRIN Leaf RESIN SeedRUTIN Leaf SALANNIN Seed SALANNOLIDE Plant SCOPOLETIN Plant STEARIC-ACIDFruit SUGIOL Bark SULFUR Seed TANNIN Stem Bark VEPININ Seed VILASANINLeaf

1. A method for extracting azadirachtin from an azadirachtin-containingsample, comprising: (a) dissolving said sample in an organic polarsolvent; (b) combining the solution of step (a) with an organicnon-polar solvent to form a single phase containing the said solventsand dissolved sample; (c) treating said single phase of step (b) with anaqueous salt solution to form an aqueous phase and a phase containingthe organic non-polar solvent; (d) recovering the aqueous phase; and (e)treating the recovered aqueous phase to isolate an azadirachtin Acomponent therefrom.
 2. A method according to claim 1, wherein both theaqueous phase and the organic non-polar phases are recovered and therecovered organic non-polar phase treated in accordance with steps (c)and (d) of claim
 1. 3. A method according to claim 2, wherein therecycling of the organic non-polar phase is carried out a plurality oftimes.
 4. A method according to claim 3, wherein the aqueous phase ineach cycle is recovered and then pooled with each other prior toisolation of a solid product therefrom.
 5. A method according to claim1, wherein, the organic polar solvent in step (a) is selected fromacetone, methanol and ethanol or mixtures thereof.
 6. A method accordingto claim 1, wherein the organic non-polar solvent used in step (b) is ahalogenated hydrocarbon.
 7. A method according to claim 6, wherein theorganic non-polar solvent is toluene, carbon tetrachloride, chloroformor dichloromethane.
 8. A method according to claim 1, wherein the singlephase of step (b) is treated with a non-saturated aqueous salt solution.9. A method according to claim 1, wherein the aqueous salt solution is asolution of a salt which forms a solution which is approximately neutralin pH.
 10. A method according to claim 9, wherein the salt is selectedfrom water soluble alkali metal and alkaline earth metal halides.
 11. Amethod according to claim 10, wherein the salt is sodium chloride.
 12. Amethod according to claim 1, wherein the salt solution contains at leastabout 5% salt by weight.
 13. A method according to claim 1, wherein theorganic non-polar phase is recovered and treated to isolate anazadirachtin B component.
 14. A method according to claim 2, wherein theorganic non-polar phase, after further treatment in accordance withsteps (c) and (d), is recovered and treated to isolate an azadirachtin Bcomponent.
 15. A method for extracting azadirachtin from anazadirachtin-containing sample, comprising: (a) dissolving said samplein an organic polar solvent; (b) combining the solution of step (a) withan organic non-polar solvent to form a single phase containing the saidsolvents and dissolved sample; (c) treating said single phase of step(b) with an aqueous salt solution to from an aqueous phase and a phasecontaining the organic non-polar solvent; (d) recovering the phasecontaining the organic non-polar solvent; (e) treating the recoveredorganic non-polar phase to isolate an azadirachtin B componenttherefrom.
 16. A method according to claim 15, further comprisingrecovering the aqueous phase.
 17. A method according to claim 15,further comprising treating the recovered organic non-polar phase inaccordance with steps (c) and (d) prior to step (e).
 18. A methodaccording to claim 17, wherein the treating of the organic non-polarphase in accordance with steps (c) and (d) is carried out a plurality oftimes.